| Citation: |
BI Xiaomin, HUANG Guiju, FAN Sigang, ZHU Wenjie, YU Dahui. Optimization of expression conditions for recombinant protein of biomineral gene Pearlin from pearl oyster (Pinctada fucata)[J]. South China Fisheries Science, 2015, 11(6): 100-106. DOI: 10.3969/j.issn.2095-0780.2015.06.014
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To obtain high and stable expression recombinant fusion protein of Pearlin, we constructed the recombinant vector by using the open reading frames (ORF) of Pearlin cloned from the mantle tissue of pearl oyster (Pinctada fucata) and optimized the expression conditions. The Pearlin ORF was cloned into the vector pET32a and the plasmids of pET32a-Pearlin were obtained and then transformed into E.coli BL21(DE3). His-tagged insoluble fusion protein was highly expressed and the molecular weight of the fusion protein was about 34.19 kD. We optimized the conditions for IPTG concentrations, induction duration and timing, and temperature and pH of the medium. The results showed that the optimal IPTG concentration ranged from 0.6 mmol·L-1 to 1.4 mmol·L-1 and the best temperature was 37 ℃ when IPTG concentration was 1.0 mmol·L-1 and incubation time was 6 h. The expression levels of recombinant protein did not change significantly when the pH of medium was 6.0, 7.0 and 8.0. When IPTG concentration was kept constant (1.0 mmol·L-1), 4~6 h induction was optimal; when IPTG concentration (1.0 mmol·L-1) and induction duration (6 h) were kept constant, the best starting time for induction was 3~4 h after transformation. Solubility test indicates that fusion protein pET32a-Pearlin was mainly in the form of inclusion body.
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